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1.
Genes Brain Behav ; 17(8): e12516, 2018 11.
Article in English | MEDLINE | ID: mdl-30133126

ABSTRACT

Adhesion molecules of the immunoglobulin superfamily (IgSF) are essential for neuronal synapse development across evolution and control various aspects of synapse formation and maturation. Neph2, also known as Kirrel3, is an IgSF adhesion molecule implicated in synapse formation, synaptic transmission and ultrastructure. In humans, defects in the NEPH2 gene have been associated with neurodevelopmental disorders such as Jacobsen syndrome, intellectual disability, and autism-spectrum disorders. However, the precise role in development and function of the nervous system is still unclear. Here, we present the histomorphological and phenotypical analysis of a constitutive Neph2-knockout mouse line. Knockout mice display defects in auditory sensory processing, motor skills, and hyperactivity in the home-cage analysis. Olfactory, memory and metabolic testing did not differ from controls. Despite the wide-spread expression of Neph2 in various brain areas, no gross anatomic defects could be observed. Neph2 protein could be located at the cerebellar pinceaux. It interacted with the pinceau core component neurofascin and other synaptic proteins thus suggesting a possible role in cerebellar synapse formation and circuit assembly. Our results suggest that Neph2/Kirrel3 acts on the synaptic ultrastructural level and neuronal wiring rather than on ontogenetic events affecting macroscopic structure. Neph2-knockout mice may provide a valuable rodent model for research on autism spectrum diseases and neurodevelopmental disorders.


Subject(s)
Membrane Proteins/genetics , Membrane Proteins/physiology , Animals , Carrier Proteins/genetics , Cell Adhesion/physiology , Immunoglobulins/physiology , Mice , Mice, Knockout , Neurogenesis , Neurons/metabolism , Synapses/metabolism
2.
EMBO Rep ; 18(9): 1521-1535, 2017 09.
Article in English | MEDLINE | ID: mdl-28710093

ABSTRACT

Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.


Subject(s)
Actins/metabolism , Cilia/metabolism , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Proteome/metabolism , Actins/chemistry , Animals , Computational Biology , Gene Expression Regulation , Gene Knockout Techniques , Humans , Membranes/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Myosins/deficiency , Myosins/genetics , Myosins/metabolism , Proteomics , Signal Transduction
3.
Sci Signal ; 10(474)2017 Apr 11.
Article in English | MEDLINE | ID: mdl-28400537

ABSTRACT

Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target CTGF was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Mechanotransduction, Cellular , Phosphoproteins/metabolism , Podocytes/metabolism , Transcription Factors/metabolism , Acyltransferases , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Fluorescent Antibody Technique , HEK293 Cells , Humans , Kidney Glomerulus/metabolism , Male , Mice , Phosphoproteins/genetics , Podocytes/cytology , Podocytes/drug effects , Proteinuria/genetics , Proteinuria/metabolism , Proteomics , Puromycin Aminonucleoside/pharmacology , Rats , Stress, Mechanical , Transcription Factors/genetics , YAP-Signaling Proteins
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